METHOD AND ITS COMPOSITIONS FOR DETECTION OF NUCLEIC ACID TARGET FROM BIOLOGICAL SAMPLES AND BODY FLUIDS

Abstract

Current invention is directed for rapid sample pretreatment method that allows highly sensitive and specific detection of target nucleic acid (eg human genomic DNA, human pathogen genomic DNA, human non-pathogen genomic DNA) by amplification directly from crude unpurified biological samples lysates (eg human urine, saliva, blood, urethra and cervical swabs and other samples containing biological material). Invention is focused on the description of the biological sample pretreatment method that enables fast release of the genomic material from human and pathogen cells, components of what are compatible with the following nucleic acid amplification method. As an example of the application, invention also discloses protocols and primer sequences for isothermal nucleic acid amplification (recombinase polymerase amplification--RPA, loop-mediated isothermal amplification--LAMP), that enable highly specific and sensitive diagnostics of the genomic material from Homo sapiens, Chlamydia trachomatis and Mycoplasma genitalium from crude biological sample lysates and/or purified total DNA. The example amplification can be combined with immunochromotographic product detection using lateral-flow strips and allows rapid (under 20 min) isothermal nucleic acid amplification based C. trachomatis and M. genitalium diagnostics from human urine samples, that does not require specific laboratory equipment nor qualified personnel, and is therefore well suited for point-of-care settings applications.

Description

PRIORITY

[0001] This application is a national entry of PCT/EP2013/071906 filed on Oct. 20, 2013 and claiming claims priority of U.S. 61/616,495 filed on Jun. 4, 2010, both of which are fully incorporated herein by reference.

SEQUENCE LISTING

[0002] This application contains sequence data provided on a computer readable diskette and as a paper version. The paper version of the sequence data is identical to the data provided on the diskette.

FIELD OF THE INVENTION

[0003] The invention is directed to compositions and method for rapid biological sample pretreatment that allows following nucleic acid amplification based detection of the target nucleic acid from biological samples and body fluids.

BACKGROUND OF THE INVENTION

[0004] Current diagnostics relies majorly on the nucleic acid amplification techniques (NAAT). Most commonly known method for specific DNA amplification is PCR that gives reasonable sensitivity on the laboratory level. Lately new emerging techniques have been developed of isothermal amplification, such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), helicase dependent amplification (HDA). These isothermal NAATs do not require thrermocycling of the reaction and have shown extremely high levels of sensitivity, resulting in detectable amplification product from as few as 1-2 template copies. Isothermal reaction makes them well suited for point-of-care (POC) settings (eg GP office, at home), bringing diagnostics test conveniently and immediately to the patient and decreasing time to result. In the field of sexually transmitted diseases, POC diagnostics also allows private and non-invasive testing, that has a potential to significantly reduce the spread of the pathogens, especially those that exist in asymptomatic form like C. trachomatis and M. genitaium.

[0005] Both M. genitalium and C. trachomatis infections are known as "silent" diseases as they often remain asymptomatic. Thus regular diagnostic screening of these sexually transmitted pathogens is of high importance. Classically C. trachomatis infection has been diagnosed from urethral or cervical swab specimens by tissue culture method. Because culturing identifies only viable C. trachomatis cells, sensitivity of the diagnostics is affected by the freshness of the specimen depending on the time between collection and processing in the laboratory. Thus during 1980s antigen and nucleic acid detection technologies have been developed for C. trachomatis diagnostics that have lesser demand of cost, time, expertise, preservation of infectivity during transport. Furthermore nucleic acid detection techniques have proved to have much higher sensitivity levels as they can detect pathogen DNA from unviable cells or cell debris. Microbiological detection of M. genitalium is also mostly performed by specific amplification of the pathogen DNA by PCR. M. genitalium culture is extremely difficult and is not performed routinely. Serological detection methods of M. genitalium are weakly sensitive and specific.

[0006] Although NAAT open up crucial opportunity for highly effective diagnostics, to date they are routinely used only on the laboratory level. NAATs are complicated to perform, require trained personnel and expensive machinery. Thus NAAT based diagnostics is centered to large hospitals and diagnostics centers. One of the major limitations of the NAAT techniques is the requirement for pure DNA sample. The purity of the sample can affect significantly performance of the NAAT-s, especially PCR. Novel isothermal NAAT-s like RPA, LAMP, HDA etc seem to be less sensitive towards nucleic acid sample purity and are able to efficiency amplify DNA present in eg human urine samples.

[0007] Current invention discloses a method and its compounds for biological sample pretreatment that allows efficient release of the genomic DNA from cellular material. Described sample pretreatment method is compatible with the following nucleic acid amplification procedure allowing detection of the target DNA from crude sample lysates. The invention allows skipping of the DNA purification step prior to NAAT analysis, having therefore an important impact on the complexity and speed of the diagnostic technique. Current invention facilitates significantly implementation of the highly sensitive and specific NAAT diagnostics in the POC settings.

[0008] Because examples of the invention implementation is concentrated on human sexually transmitted pathogen diagnostics, the overview of the Chlamydia trachomatis and Mycoplasma genitalium will be given hereafter.

[0009] C. trachomatis and M. genitalium are sexually transmitted human pathogens. Both of them are associated with non-gonococcal (non-specific) urethritis in men and several inflammatory reproductive tract syndromes in women such as cervicitis and pelvic inflammatory disease. Inflammatory diseases caused by acute untreated infections of C. trachomatis and M. genitalium are one of the leading causes of female infertility worldwide.

[0010] The prevalence of M. genitalium ranges globally from 1-4% in men and 1-6% in women. Reported prevalence data within populations at higher risk (eg within sexually transmitted disease (STD) testing centers) reach 38%. C. trachomatis prevalence rates among sexually active young people vary from 5-10% depending on the age, ethnic origin etc. C. trachomatis infection is almost always more prevalent among women and has shown an increasing trend globally during past decades.

[0011] M. genitalium is a small (0.2-0.3 μm) pleomorphic bacterium that lacks cell wall making it resistant to common antibiotics targeting cell wall (eg penicillin). M. genitalium cells are flask shaped and carry a specific adhesion organelle that allows bacteria to adhere to various materials and cells including human epithelial cells. Adhesion is the main mechanism of M. genitalium pathogenesis that involves at least seven adhesins including major adhesin MgPa (encoded by MGPB gene).

[0012] C. trachomatis is a gram-negative, obligate intracellular pathogen that has a unique biphasic developmental cycle during which they exist in two developmental forms: the EB (or elementary body) and RB (or reticulate body). EB is smaller (0.2 μM), metabolically inactive, infectious extracellular form of the organism and RB is larger (0.8 μM) metabolically active intracellular form. Chlamydial infection involves attachment of the EB to a host cell and its subsequent internalization into a membrane-bound vesicle. Inclusion differentiates into RB which uses host cell ATP and metabolites to undergo 8-12 round of cell division. RB differentiates and matures into infectious EB that are released by host cell lysis. C. trachomatis strains are serologically classified into 15 serovars based on antigenic variation of the major outer membrane protein. A-C serovars are eye pathogens causing ocular trachoma. Serovars D-K and L1-L2 are sexually transmitted pathogens that infect columnar epithelial cells of the genital tract.

[0013] Adaptive immunity against C. trachomatis involves INF-γ mediated host cell responce that deprives chlamydial RBs of tryptophan, which ultimately prevents their growth and replicative capabilities. C. trachomatis genital serovars have retained some of the eubacterial tryptophan biosynthesis genes, TRPA and TRPB encoding α and β subunits of the tryptophan synthase that catalyzes conversion of the indole into tryptophan. Thus genital C. trachomatis serovars have retained the capacity to use exogenous indole secreted by genital trakt normal microflora that allows them to overcome INF-γ mediated growth restriction and promotes long term establishment of the infection.

[0014] M. genitalium has a small AT rich (68%) 0.58 Mb genome that encodes 485 genes. Despite its small size, 4% of the genome consists of repeated elements (MgPa repeats) that present homology with the MGPB gene. C. trachomatis also carries a small genome of approximately 1 Mb chromosome and 7.5 kb cryptic plasmid. Almost all C. trachomatis strains harbor four to ten plasmid copies per chromosome. Although some plasmid-free C. trachomatis isolates have been described, their virulence is significantly reduced as compared to the plasmid carrying strains. Chlamydia plasmid sequence is highly conserved (<1% variation) and contains eight major coding sequences (CDSs) along with a replication origin formed by four 22 bp tandem repeats. In silico analysis has identified plasmid encoded proteins to have a function in replication.

DESCRIPTION OF THE INVENTION

[0015] Current invention discloses a method and its compounds for biological sample pretreatment that allows efficient release of the genomic DNA from cellular material. Major advantage of the described sample pretreatment method is its compatibility with downstream nucleic acid amplification procedures allowing detection of the target DNA from crude sample lysates. Thus current invention allows skipping of the DNA purification step prior to NAAT analysis, having therefore an important impact on the complexity and speed of the diagnostic technique.

[0016] The invention discloses cell lytic compounds that allow fast (within 5 min at RT° C.) and efficient release of the genomic material from mammalian cells, their pathogen and commensal microorganisms, bacterial and fungi cultures etc. Sample pretreatment buffer consists of membrane active (cell-penetrating) peptides, mild detergents or a combination of the above two.

[0017] Membrane active peptides have antibacterial and antimicrobial effect acting disruptively on bacterial membranes. They are also known as cell membrane penetrating agents that can deliver different cargo molecules into mammalian cells (eg oligonucleotides, siRNA, plasmids, peptides). Current invention targets novel usage of the cell-penetrating peptides for diagnostics purposes. At higher (μM-mM) concentrations cell-penetrating peptides disrupt cellular membranes, that allows the release of the genomic DNA that can be used as a target in the following nucleic acid amplification reaction. Cell membrane disruptive peptides have shown no or minimal inhibiting effect on nucleic acid amplification even at high concentrations, thus can be efficiently used as agents facilitating genomic material release.

[0018] Detergents are very good solubilizing agents, but they tend to denature proteins by destroying native three dimensional structures. Certain combination of the mild ionic or non-ionic detergents (eg Triton X-100, Triton X-114, NP-40, CHAPS, Octyl-β-glucoside, Octyl-β-thioglucopyronoside) at low (eg 0.1-1%) concentration allow efficient cell wall disruption in order to release genomic material enclosed within cells. These mild detergents do not interfere significantly with nucleic acid amplification procedure, and are able to induce or facilitate the release of the sufficient amount of the target nucleic acid. The composition and concentration of the detergents is set to efficiently lyse cells within 5 min RT° C. incubation.

[0019] The ability of the membrane active peptide and/or detergent mediated sample pretreatment to convert biological sample into material well usable for the nucleic acid amplification is the major focus of the invention and has been confirmed by establishing detection of the Chlamydia trachomatis, Mycoplasma genitalium and Homo sapiens genomic DNA from crude human urine lysates.

[0020] For that a diagnostic method for highly specific and sensitive C. trachomatis and M. genitalium detection from human samples has been developed based on isothermal nucleic acid amplification (RPA, LAMP) and including immunochromotographic product detection using lateral-flow strips. For both pathogens we have used double target system, where simultaneous detection of two different genomic targets is performed. This reduces probability of the false negative diagnostics test result in case deletions or mutations are introduced into pathogen genomic DNA regions used as the amplification targets. All target regions were selected based on their high homology among different pathogen strains and lack of identity with similar species.

[0021] For C. trachomatis detection we have used genomic sequence regions from a well-established diagnostic target--coding sequence 2 of the multicopy cryptic plasmid (CDS2). For the second target we have chosen β subunit of the tryptophan synthase gene TRPB. For M. genitalium detection we have used genomic sequence regions from gene encoding MgPa dominant adhesin (MGPB) that is the main component of multiple repeats throughout its genome. For the second target we used 16S rRNA gene that is also present in multiple copies within M. genitalium genome. M. genitalium 16S rRNA gene however is highly conserved between different Mycoplasma species (eg 98% identity with M. pneumoniae, 91% with M. gallisepticum). Thus multiple mutations containing regions were chosen for the isothermal amplification and additional specificity testing was performed for this particular target.

[0022] For each target, optimal primer pair combinations were established that enable highest sensitivity levels for the assay. Optimized RPA reaction allowed well detectable and stable product amplification with minimum of 20-50 target sequence copies. Optimized LAMP reaction with loop primers allowed product amplification with minimum of 5-10 target sequence copies. Each diagnostics target was tested for specificity of the reaction with 50 000 copies (0.16 ng) of H. sapiens genomic DNA and in case of M. genitalium 16S rRNA target also with 100 000 copies of M. pneumoniae genomic DNA. Isothermal amplification sensitivity and specificity was verified with total DNA extracted from human urine samples.

[0023] Major objective of the current invention was to develop a diagnostic assay applicable under point-of-care conditions. Thus we have integrated immunochromotographic amplification product detection into the diagnostics system. For that purpose, forward primer sequences were 5' labeled with biotin and reverse primers with fluorescein amidite (FAM). During amplification reaction a dually labeled products were produced, that were detected within minutes using lateral-flow strips. Integration of the immunochromotographic product detection required additional primer optimization. Primers gaining template independent lateral-flow strip detectable signal were eliminated from the selection.

[0024] RPA and LAMP isothermal amplification based diagnostics methods were also showed to be suitable for simultaneous multiple target detection. Both assays were optimized for H. sapiens GAPDH gene target to be used as a positive control of the diagnostics test with human samples. PCR and isothermal amplification (RPA/LAMP/HDA) protocols were adjusted for optimal sensitivity and high specificity of the diagnostics test.

[0025] The present method for detection of nucleic acid target(s) from biological crude samples and body fluids comprises following steps:

[0026] a) sample pretreatment comprising cell lysis and release of nucleic acid targets in biological samples and body fluids such as tissue, urine, saliva, blood, stool, hair, etc. and their derivatives, but not limited to the examples list, wherein the lytic peptides are used to release nucleic acid targets in biological samples;

[0027] b) amplification of nucleic acid(s) comprising nucleic acid, such as DNA, RNA and their derivatives but not limited to the list, amplification initiated by presence of target and comprise amplification methods such as PCR (Polymerase Chain Reaction), HCR (Hybridization Chain Reaction), RCA (Rolling Circle Amplification), RPA (Recombinase Polymerase Amplification), LAMP (Loop mediated isothermal AMPlification), HDA (Helicase Dependent Amplification), etc. and their derivatives, but not limited to the examples list, wherein one or more specific target based sequences are amplified or sample solution obtained during the step (1) is directly subjected for further amplification procedure;

[0028] c) detection of amplification product(s) comprising the use of qualitative or quantitative detection methods such as sandwich assays, ELISAs (Enzyme Linked ImmunoSorbent Assay), LF (Lateral Flow) immunochromatographic assays, wavelength changing (visible spectrum, chemiluminescence, fluorescence, phosphorescence and etc.) dyes, denrimeres, etc. or corresponding moiety conjugated detector molecules and ligands, with or without optical apparatus, appropriate wavelength emitter or reader or their combination, wherein qualitative and quantitative detection is performed with crude sample solution.

[0029] The pretreatment method is specifically designed to detect nucleic acid target(s):

[0030] of Chlamydia trachomatis with the use of specific target region provided in Table 1 or with the use of specific primer(s) and/or its labeled derivative(s) sequences provided in Table 2, 3; and

[0031] Mycoplasma genitalium with the use of specific target region provided in Table 1 or with the use of specific primer(s) and/or its labeled derivative(s) sequences provided in Table 2, 3.

[0032] The present method with human genomic GAPDH target is used for detection:

[0033] as an internal validation and platform assessing technique;

[0034] as an internal validation and platform assessing technique with specific primer(s) and/or its labeled derivative(s) sequences provided in Tables 2, 3.

[0035] The pretreatment method that relates to molecular diagnostics of Chlamydia trachomatis wherein TRPB gene is used as molecular diagnostics target.

EXAMPLES OF THE IMPLEMENTATION

Example 1

Fast Diagnostics of the Presence of Chlamydia trachomatis in a Urine Sample

[0036] Present protocol describes method and its components for highly sensitive Chlamydia trachomatis diagnostics from human urine sample. The whole procedure including sample pretreatment, target isothermal amplification and product detection takes under 20 min and requires 10 min incubation at 37° C. Described method detects two C. trachomatis targets TRPB sequence in the genomic region and CDS2 sequence in the cryptic plasmid region (Table 1).

TABLE-US-00001 TABLE 1 Genomic regions of Chlamydia trachomatis, Mycoplasma genitalium and Homo sapiens used for isothermal amplification based detection Target organism Sequence name Genebank accession nr C. trachomatis PL-CDS2 FM865439.1 sequence 756-1748 TRPB FN652779.2 sequence 193461-194639 M. genitalium 16S rRNA CP003773.1 sequence 169843-171366 MGPA CP003773.1 sequence 221365-225744 H. sapiens GAPDH NG_007073.2

[0037] Both of the C. trachomatis targets are amplified using highly specific and sensitive primers that carry same labeling, forward primers are labeled with biotin and reverse with FAM. Thus C. trachomatis specific products are not distinguished during immunochromatographic detection on lateral-flow strips. Detection of the two C. trachomatis regions is used to ensure positive test results in case one of the target regions is mutated or deleted. The reaction also contains primers targeting H. sapiens GAPDH gene that produce DIG and FAM labeled product. This product is recognized as a separate lane on the lateral-flow strip and serves as a positive control for the whole procedure (release of the genomic material from cells, amplification and detection). Analytical sensitivity of the described method is 50 C. trachomatis cells and 50 H. sapiens cells per test. This allows detection of the C. trachomatis in the first void urine at pathogen concentration of 10 000 cells per 1 ml of urine or higher.

[0038] Patient urine sample is mixed with equal volume of sample pretreatment buffer containing 0.2% Triton X-114, 150 mM NaCl, 50 mM Tris pH 7.0, and incubated 5 min at RT° C. 10 μl of the treated sample is used in the RPA reaction containing following components: C. trachomatis PL-CDS2 5' biotin labeled FW3 primer at 0.4 μM final concentration, C. trachomatis PL-CDS2 5' FAM labeled RV1 primer at 0.4 μM final concentration, C. trachomatis TRPB 5' biotin labeled FW2 primer at 0.4 μM final concentration, C. trachomatis TRPB 5' FAM labeled RV3 primer at 0.4 μM final concentration, H. sapiens GAPDH 5' DIG labeled FW3 primer at 0.4 μM final concentration, H. sapiens GAPDH 5' FAM labeled RV2 primer at 0.4 μM final concentration (see Table 2 for primer sequences), 14 mM magnesium acetate, TwistDX RPA enzyme pellet and 29.5 μl of the rehydration buffer. Reaction is incubated at 37° C. for 10 min. The products are diluted 1:10 ratio with dilution buffer and analyzed on lateral-flow strips detecting Biotin-FAM and DIG-FAM labeled molecules.

TABLE-US-00002 TABLE 2 Specific primer sequences for recombinase polymerase amplification (RPA) against targets provided in Table 1 Target SEQ organism ID and region Sequence (5'-3') NO C. trachomatis Forward FW1 5'- CTTCTTTGAAGCGTTGTCTTCTCGAGAAGATTT 1 PL-CDS2 (FW) FW2 5'- CTTCTCGAGAAGATTTATCGTACGCAAATATC 2 primer FW3 5'- 3 sequences CCTTCATTATGTCGGAGTCTGAGCACCCTAGGC FW4 5'- AGGCGTTTGTACTCCGTCACAGCGGTTGCTCG 4 Reverse RV1 5'- CTCTCAAGCAGGACTACAAGCTGCAATCCCTT 5 (RV) RV2 5'- ATGGTGGGGTTAAGGCAAATCGCCCGCACGTT 6 primer RV3 5'- TCT TCG TAA CTC GCT CCG GAA AAA TGG 7 sequences TGG GG RV4 5'- CTT TCT ACA AGA GTA CAT CGG TCA ACG AAG 8 AGG C. trachomatis Forward FW1 5'- ACT ATG CGG GGA GAC AAA CTC CTC TGA 9 TRPB (FW) CTG AAG primer FW2 5'- TCT TAA ACG CGA AGA TCT TTT GCA TAC AGG 10 sequences AGC FW3 5'- CAT ACA GGA GCA CAT AAA CTG AAT AAT GCT 11 CTT GG FW4 5'- CTC TTG GTC AGT GTT TGC TTG CTA AAT ATC 12 TTG Reverse RV1 5'- TCC CGC ACC TGT TTC AGC TAC AAC ACG TGT 13 (RV) TT primer RV2 5'- CTG TTG CTG TTG CTA CTC CAT GTT GTC CCG 14 sequences CAC RV3 5'- TCC CAT GTA TAC TAC ACA ATC TAA TCC TAG 15 ATA RV4 5'- TTC TGT CGT TCC ACA TCT TTT GCT CCC ATG 16 TAT M. genitalium Forward FW1 5'- AGC GCA ACC CTT ATC GTT AGT TAC ATT GTT 17 16S rRNA (FW) TAA primer FW2 5'- CGT TAG TTA CAT TGT TTA ACG AGA CTG CTA 18 sequences ATG T FW3 5'- ACG TGC TAC AAT GGC CAA TAC AAA CAG 19 TAG CCA A Reverse RV1 5'- TTG CAG CCC TCA ATC CGA ACT GAG ACC 20 (RV) AAC TTT T primer RV2 5'- CAT AGC TGA TTC GCG ATT ACT AGT GAT TCC 21 sequences AGC RV3 5'- TTC CAA TAA AGG TTA GCA ACA CGT TTT TAA 22 ATA M. genitalium Forward FW1 5'- TTGGACTTGAAACAATAACAACTTCTCTTCACT 23 MGPA (FW) FW2 5'- 24 primer AAGATTACTGGAGAGAACCCAGGATCATTTGGA sequences FW3 5'- CAG TGG GCA GAC TAT GTC TTA CCT TTG ATT 25 GTA FW4 5'- TTA TCC TTA GTG TTA CTT TGG GAT TAA CGA 26 TTG G FW5 5'- 27 CAATGCACAGAAACAAAAAGGCATTACAAGCAGGG Reverse RV1 5'- TCT GAT TGC AAA GTT TTG CTG ACC ATC AAG 28 (RV) GTA primer RV2 5'- CTC TAC CGT TGT TAT CAT ACC TTC TGA TTG 29 sequences C RV3 5'- TTC TGT TAA TGA TCT CTT TAA AGA CAC TAC 30 CAA RV4 5'- CTT AGG AGC GTT AGA GAT CCC TGT TCT GTT 31 AAT G RV5 5'- CTT GTT TTA ACT TCT TAG GAG CGT TAG AGA 32 TCC C RV6 5'- 33 TTACTGGAGGTTTTGGTGGGGTTTTAGGAGTTGG H. sapiens Forward FW1 5'- 34 GAPDH (FW) CTCCTCCGGGTGATGCTTTTCCTAGATTATTCTC primer FW2 5'- CTA ACC CTG CGC TCC TGC CTC GAT GGG 35 sequences TGG AG FW3 5'- AAG TCA GGT GGA GCG AGG CTA GCT GGC 36 CCG ATT Reverse RV1 5'- TCC TTT TCC AAC TAC CCA TGA CTC AGC TTC 37 (RV) TCC C primer RV2 5'- CAC CAT GCC ACA GCC ACC ACA CCT CTG 38 sequences CGG GGA RV3 5'- CCA CCA CCA GAG GGG CCA TTT TGC GGT 39 GGA AAT

[0039] Chlamydia tests positive if the test gives 2 lines (Biotin-FAM and DIG-FAM), negative if the test gives 1 line DIG-FAM. The results of the test are invalid if none of the lines are present or only Biotin-FAM line is present.

Example 2

Fast Diagnostics of the Presence of Mycoplasma genitalium in a Urine Sample

[0040] Present protocol describes method and its components for highly sensitive Mycoplasma genitalium diagnostics from human urine sample. The whole procedure including sample pretreatment, target isothermal amplification and product detection takes under 20 min and requires 10 min incubation at 37° C. Described method detects two M. genitalium targets MGPA and 16S rRNA sequences in the pathogen genome (Table 1). Both of the M. genitalium targets are amplified using highly specific and sensitive primers that carry same labeling, forward primers are labeled with biotin and reverse with FAM. Thus M. genitalium specific products are not distinguished during immunochromatographic detection on lateral-flow strips.

[0041] Detection of the two M. genitalium regions is used to ensure positive test results in case one of the target regions is mutated or deleted. The reaction also contains primers targeting H. sapiens GAPDH gene that produce DIG and FAM labeled product. This product is recognized as a separate lane on the lateral-flow strip and serves as a positive control for the whole procedure (release of the genomic material from cells, amplification and detection). Analytical sensitivity of the described method is at least 50 M. genitalium cells and 50 H. sapiens cells per test. This allows detection of the M. genitalium in the first void urine at pathogen concentration of 10 000 cells per 1 ml of urine or higher.

[0042] Patient urine sample is mixed with equal volume of sample pretreatment buffer containing 0.2% NP-40, 150 mM NaCl, 50 mM Tris pH 7.0, and incubated 5 min at RT° C. 10 μl of the treated sample is used in the RPA reaction containing following components: M. genitalium MGPA 5' biotin labeled FW4 primer at 0.4 μM final concentration, M. genitalium MGPA 5' FAM labeled RV4 primer at 0.4 μM final concentration, M. genitalium 16S rRNA 5' biotin labeled FW1 primer at 0.4 μM final concentration, M. genitalium 16S rRNA 5' FAM labeled RV1 primer at 0.4 μM final concentration, H. sapiens GAPDH 5' DIG labeled FW3 primer at 0.4 μM final concentration, H. sapiens GAPDH 5' FAM labeled RV2 primer at 0.4 μM final concentration (see Table 2 for primer sequences), 14 mM magnesium acetate, TwistDX RPA enzyme pellet and 29.5 μl of the rehydration buffer. Reaction is incubated at 37° C. for 10 min. The products are diluted 1:10 ratio with dilution buffer and analyzed on lateral-flow strips detecting Biotin-FAM and DIG-FAM labeled molecules.

[0043] M. genitalium tests positive if the test gives 2 lines (Biotin-FAM and DIG-FAM), negative if the test gives 1 line DIG-FAM. The results of the test are invalid if none of the lines are present or only Biotin-FAM line is present

Example 3

Highly Sensitive Diagnostics of the Presence of Chlamydia trachomatis from a Patient Sample Extracted Total DNA

[0044] Present method uses highly sensitive loop mediated isothermal amplification (LAMP) for specific detection of C. trachomatis DNA. Analytical sensitivity of the described method is at least 5 C. trachomatis cells per test. LAMP reaction is prepared as follows: C. trachomatis PL-CDS2 SET4 primers F3 and B3 at 0.2 μM concentration each, C. trachomatis PL-CDS2 SET4 5' biotin labeled FIP and 5' FAM labeled BIP primers at 1.6 μM each, C. trachomatis PL-CDS2 SET4 5' biotin labeled LF and 5' FAM labeled LB loop primers at 0.8 μM each (see Table 3 for primer sequences), 5.6 μM dNTP, 6 mM MgSO₄, 0.8 M betain, 8 units of Bst polymerase, 2.5 μl of 10× Bst polymerase buffer and 5 μl of total DNA extracted from patient sample per 25 μl reaction. Incubate reaction for 1 h at 63° C., dilute diluted 1:10 ratio with dilution buffer and analyzed on lateral-flow strips detecting Biotin-FAM labeled molecules.

[0045] In a parallel reaction C. trachomatis TRPB targeting LAMP can be performed with SET1 primers (Table 3) for additional positive control (with analytical sensitivity of at least 5 C. trachomatis cells per test). Additionally H. sapiens GAPDH targeting LAMP with SET 1 primers (Table 3) could be used as a positive control of the reaction.

TABLE-US-00003 TABLE 3 Specific primer sequences for loop mediated isothermal amplification (LAMP) against targets provided in Table 1 Target SEQ organism and ID region Sequence (5'-3') NO: C. trachomatis SET F3 GCTTGTTGGAAACAAATCTGA 40 PL-CDS2 1 B3 TCGAACATTTTTTAAAACCAGG 41 FIP GATCGCCCAGACAATGCTCCTAATCTCCAAGCTTAAGACTTCA 42 BIP AACCAATCCCGGGCATTGATAAAAACGGATGCGATGAAC 43 SET F3 AAAGTGCATAAACTTCTGAGG 44 2 B3 CTAAAAAAAATCAATGCCCGG 45 FIP TGTTTCCAACAAGCTACCATTTCTTATAATCCTCTTTTCTGTCTGACG 46 BIP AATCTCCAAGCTTAAGACTTCAGAGATTGGTTGATCGCCCAGA 47 SET F3 TCTAAAGACAAAAAAGATCCTCG 48 3 B3 TGTGATGGGTAAAGGGATT 49 FIP GCATGAAAAGCTTCTCCTTATTCGAATGATCTACAAGTATGTTTGTTGAG 50 BIP CCAATAGGATTCTTGGCGAATTTTTTGCAGCAAGAAATGTCGTTA 51 SET F3 CGACTATTTTCTTGTTTAGAAGGTT 52 4 B3 GAAAAGATTGGTCTATTGTCCT 53 FIP AGCAGCAAGCTATATTTCCTTAACAGCTATAGCGACTATTCCTTGA 54 BIP GTCTTGGCAGAGGAAACTTTTTTAATGGATATGAATCTGCAAGAGTT 55 LF1 GATTCCTAAACAGGATGAC 56 LB1 TCGCATCTAGGATTAGAT 57 LF3 AGATTCCTAAACAGGATGAC 58 LB2 CGCATCTAGGATTAGATTATG 59 SET F3 AATATCATCTTTGCGGTTGC 60 5 B3 TCTACAAGAGTACATCGGTCA 61 FIP TCGAGCAACCGCTGTGACGACCTTCATTATGTCGGAGTC 62 BIP GCAGCTTGTAGTCCTGCTTGAGTCTTCGTAACTCGCTCC 63 LF TAC AAA CGC CTA GGG TGC 64 LB CGG GCG ATT TGC CTT AAC 65 C. trachomatis SET F3 GCA GTT GCA GGA AGA GAT C 66 TRPB 1 B3 GTC ATC TTG AAG AAG ATA CGA A 67 FIP GGA CTT TTG GAT TCG GGA TAA AAT GCT GAT 68 ATT CTG ATT GCA TGT ATC G BIP GGA GGA CTG GGC ATT TCT TCA TGG AAT ACT 69 CCA GGT CGC LF1 AGCGTTGGAGCCACCTC 70 LB1 GAAAACATGCAGCACGTTTTGCA 71 LF2 CAATAGCGTTGGAGCCACCT 72 LB2 AACATGCAGCACGTTTTGCA 73 SET F3 CAAGATGACGATGGACAAGT 74 2 B3 CCAGATAAGTTAACGATGACGA 75 FIP GGCTCGTCCTGACTCATGCTCCGCTGGATTAGATTATCCT 76 BIP CCGATGAAGAGGCGTTACGAGGAGCATGTGAAGA CTCCAAT 77 LF CAT GAT CTG GCC CAA CTG A 78 LB TCC TGC TTA CTA GAA ATG AGG G 79 M. genitalium SET F3 ATTGGTTAACTTACCTAGTGGC 80 MGPA 1 B3 ACTTCTTAGGAGCGTTAGAGA 81 FIP GACATAGTCTGCCCACTGGTTGATCCTCAAACCCAACAGTT 82 BIP AGGCATTACAAGCAGGGTTTGAAAGACACTACCAACTGCTT 83 LF AAAGGGTTGAAAGACAGTTTGG 84 LB AAGGTTGATGTCTTGACCA 85 F3 CACCTTACCAGTAACTGAACT 86 SET B3 AACCCTGCTTGTAATGCC 87 2 FIP TTAAGCGGATTGAAGCTTGATCTGTCTATGACCAGTATGTACCA 88 BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 89 LF GCCACTAGGTAAGTTAACCAAT 90 LB AATGCATCAAGTACAGGTCC 91 SET F3 CACCTTACCAGTAACTGAACT 92 3 B3 AACCCTGCTTGTAATGCC 93 FIP TTAAGCGGATTGAAGCTTGATCTCTATGACCAGTATGTACCACT 94 BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 95 LF GCCACTAGGTAAGTTAACCAAT 96 LB AATGCATCAAGTACAGGTCC 97 SET F3 CACCTTACCAGTAACTGAACT 98 4 B3 AACCCTGCTTGTAATGCC 99 FIP TTAAGCGGATTGAAGCTTGATCGTCTATGACCAGTATGTACCAC 100 BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 101 LF GCCACTAGGTAAGTTAACCAAT 102 LB AATGCATCAAGTACAGGTCC 203 SET F3 GATCCTCAAACCCAACAGTT 104 5 B3 TTAGGAGTTGGTTTGGTTGG 105 FIP GACATAGTCTGCCCACTGGTTTGCATCAAGTACAGGTCC 106 BIP AGGCATTACAAGCAGGGTTTGAACTTCTTAGGAGCGTTAGAGA 107 LF AAAGGGTTGAAAGACAGTTTGG 108 LB AAGGTTGATGTCTTGACCAA 109 SET F3 TGTCTATGACCAGTATGTACCA 110 6 B3 AACCCTGCTTGTAATGCC 111 FIP ACTGTTGGGTTTGAGGATCTTTATTGGTTAACTTACCTAGTGGC 112 BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 113 LF TTAAGCGGATTGAAGCTTGATC 114 LB CCAAACTGTCTTTCAACCCTTT 115 SET F3 CACCTTACCAGTAACTGAACT 116 7 B3 AACCCTGCTTGTAATGCC 117 FIP TTACCTTTAAGCGGATTGAAGCTGACCAGTATGTACCACTATTG 118 BIP CCCAACAGTTTATCCCGGTACTAAGGTAAGACATAGTCTGCC 119 LF GATCAAAGCCACTAGGTAAGTT 120 LB AATGCATCAAGTACAGGTCC 121 SET F3 CTATGACCAGTATGTACCACTA 122 8 B3 AACCCTGCTTGTAATGCC 123 FIP ACTGTTGGGTTTGAGGATCTTTTTGGTTAACTTACCTAGTGGC 124 BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 125 LF TTAAGCGGATTGAAGCTTGATC 126 LB CCAAACTGTCTTTCAACCCTTT 127 SET F3 TGACCAGTATGTACCACTAT 128 9 B3 AACCCTGCTTGTAATGCC 129 FIP ACTGTTGGGTTTGAGGATCTTTTGGTTAACTTACCTAGTGGCT 130 BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 131 LF TTAAGCGGATTGAAGCTTGATC 132 LB CCAAACTGTCTTTCAACCCTTT 133 SET F3 TGACCAGTATGTACCACTATTG 134 10 B3 AACCCTGCTTGTAATGCC 135 FIP ACTGTTGGGTTTGAGGATCTTTGTTAACTTACCTAGTGGCTT 136 BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 137 LF TTAAGCGGATTGAAGCTTGATC 138 LB CCAAACTGTCTTTCAACCCTTT 139 SET F3 GACCAGTATGTACCACTATT 140 11 B3 AACCCTGCTTGTAATGCC 141 FIP ACTGTTGGGTTTGAGGATCTTTGGTTAACTTACCTAGTGGCTT 142 BIP CCCGGTACTAAATGCATCAAGTAAGGTAAGACATAGTCTGCC 143 LF TTAAGCGGATTGAAGCTTGATC 144 LB CCAAACTGTCTTTCAACCCTTT 145 M. genitalium SET F3 CGTGAACGATGAAGGTCTT 146 16S rRNA 1 B3 ACCACACTCTAGACTGATAGTT 147 FIP GCGACTGCTGGCACATAGTTAAGAATGACTCTAGCAGGCA 148 BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 149 LF GTACAGTCAAACTCCAGCCA 150 LB GGATTTATTGGGCGTAAAGCAA 151 SET F3 CGTGAACGATGAAGGTCTT 152 2 B3 ACCACACTCTAGACTGATAGTT 153 FIP GCGACTGCTGGCACATAGTAATGACTCTAGCAGGCAATG 154 BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 155 LF TGGTACAGTCAAACTCCAGC 156 LB GGATTTATTGGGCGTAAAGCAA 157 SET F3 CGTGAACGATGAAGGTCTT 158 3 B3 ACCACACTCTAGACTGATAGTT 159 FIP GCTGGCACATAGTTAGTCGTCAGAAGAATGACTCTAGCAGGC 160 BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 161 LF GTACAGTCAAACTCCAGCCA 162 LB GGATTTATTGGGCGTAAAGCAA 163 SET F3 CGTGAACGATGAAGGTCTT 164 4 B3 ACCACACTCTAGACTGATAGTT 165 FIP GCGACTGCTGGCACATAGTTAGAATGACTCTAGCAGGCAAT 166 BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 167 LF TGGTACAGTCAAACTCCAGC 168 LB GGATTTATTGGGCGTAAAGCAA 169 SET F3 CATTACTGACGCTTAGGCTT 170 5 B3 GCCAAGGATGTCAAGTCTAG 171 FIP CTTCACTACCGAAGGGATCGCCCTAGTAGTCCACACCGTAA 172 BIP GCCTGGGTAGTACATTCGCAAAACATGCTCCACCACTTG 173 LF TCCGACAGCTAGTATCTATCGT 174 LB TGAAACTCAAACGGAATTGACG 175 SET F3 CGTGAACGATGAAGGTCTT 176 6 B3 ACCACACTCTAGACTGATAGTT 177 FIP GCGACTGCTGGCACATAGTGACTCTAGCAGGCAATGG 178 BIP ACATAGGTCGCAAGCGTTATCCCTGCCTTTAACACCAGACTT 179 LF AAAGTGGTACAGTCAAACTCCA 180 LB GGATTTATTGGGCGTAAAGCAA 181 SET F3 CAAGTGGTGGAGCATGTT 182 7 B3 TCCCTTCCTTCCTCCAATT 183 FIP CGACAACCATGCACCACCTCTAGACTTGACATCCTTGGC 184 BIP CAGCTCGTGTCGTGAGATGTTTAACTAACGATAAGGGTTGCG 185 LF GTCACTCGGTTAACCTCCATT 186 LB GGTTAAGTCCCGCAACGA 187 SET F3 AATGACTCTAGCAGGCAATG 188 8 B3 ACCACACTCTAGACTGATAGTT 189 FIP CGGATAACGCTTGCGACCTTAAGTGACGACTAACTATGTGC 190 BIP AAGCGCAGGCGGATTGAACCAATGCATACAACTGTTAAGC 191 LF TGTATTACCGCGACTGCTG 192 LB AGTCTGGTGTTAAAGGCAGC 193 SET F3 AATGACTCTAGCAGGCAATG 194 9 B3 ACCACACTCTAGACTGATAGTT 195 FIP CGGATAACGCTTGCGACCTAAGTGACGACTAACTATGTGC 196 BIP AAGCGCAGGCGGATTGAACCAATGCATACAACTGTTAAGC 197 LF TGTATTACCGCGACTGCTG 198 LB AGTCTGGTGTTAAAGGCAGC 199 SET F3 CAAGTGGTGGAGCATGTT 200 10 B3 GTTTGCAGCCCTAGACATAA 201 FIP CGACACGAGCTGACGACAACCTTGGCAAAGTTATGGAAAC 202 BIP TGGGTTAAGTCCCGCAACGCCAATTTACATTAGCAGTCTCG 203 LF CATGCACCACCTGTCACT 204 LB CGCAACCCTTATCGTTAGTTAC 205 SET F3 CGCATAAGAACTTTAGTTCGC 206 11 B3 AAGACCTTCATCGTTCACG 207 FIP TAGCTACACGTCATTGCCTTGGAGGGTTCGTTATTTGATGAGG 208 BIP CACAATGGGACTGAGACACGGAGCTTTCGCTCATTGTGAA 209 LF CCTACCAACTAGCTGATATGGC 210 LB TACTCCTACGGGAGGCAG 211 H. sapiens SET F3 TGGGTGTGAACCATGAGA 212 GAPDH 1 B3 AGTCCTTCCACGATACCAA 213 FIP TCCATAGGGTGCCAGGCTGTATGACAACAGCCTCA AGAT 214 BIP CTTTCTTTGCAGCAATGCCTCCAGTTGTCATGGATGACCTTG 215 LF CTG CCT TCC TCA CCT GAT G 216 LB TGC ACC ACC AAC TGC TTA 217 SET F3 CCCCAAAGGCCAGGCT 218 2 B3 AGAAGGGATGGGAGAGAGC 219 FIP GGAATGGGGAGAAGGGCAGGTTAAATGTCACCGGGAGGATTG 220 BIP CGGAAACCAGATCTCCCACCGGCTACAGAAAGGTCAGCAGC 221 SET F3 ATCAAGTGGGGCGATGCT 222 3 B3 GGGCAGAGATGATGACCCT 223 FIP GCACTCACCCCAGCCTTCTCGCTGAGTACGTCGTGGAGT 224 BIP AAGCTGACTCAGCCCTGCAAACCCTGCAAATGAGCCTACA 225 F3 GTT GAC CCG ACC CCA AAG 226 B3 AAG GGA TGG GAG AGA GCC 227 FIP CGG AAT GGG GAG AAG GGC AGA TGT CAC CGG 228 GAG GAT TGG BIP CGG AAA CCA GAT CTC CCA CCG CCA GCT ACA 229 GAA AGG TCA GC

Sequence CWU 1

1

229133DNAartificial sequencechemically synthesized 1cttctttgaa gcgttgtctt ctcgagaaga ttt 33232DNAartificial sequencechemically synthesized 2cttctcgaga agatttatcg tacgcaaata tc 32333DNAartificial sequencechemically synthesized 3ccttcattat gtcggagtct gagcacccta ggc 33432DNAartificial sequencechemically synthesized 4aggcgtttgt actccgtcac agcggttgct cg 32532DNAartificial sequencechemically synthesized 5ctctcaagca ggactacaag ctgcaatccc tt 32632DNAartificial sequencechemically synthesized 6atggtggggt taaggcaaat cgcccgcacg tt 32732DNAartificial sequencechemically synthesized 7tcttcgtaac tcgctccgga aaaatggtgg gg 32833DNAartificial sequencechemically synthesized 8ctttctacaa gagtacatcg gtcaacgaag agg 33933DNAartificial sequencechemically synthesized 9actatgcggg gagacaaact cctctgactg aag 331033DNAartificial sequencechemically synthesized 10tcttaaacgc gaagatcttt tgcatacagg agc 331135DNAartificial sequencechemically synthesized 11catacaggag cacataaact gaataatgct cttgg 351233DNAartificial sequencechemcially synthesized 12ctcttggtca gtgtttgctt gctaaatatc ttg 331332DNAartificial sequencechemically synthesized 13tcccgcacct gtttcagcta caacacgtgt tt 321433DNAartificial sequencechemically synthesized 14ctgttgctgt tgctactcca tgttgtcccg cac 331533DNAartificial sequencechemically synthesized 15tcccatgtat actacacaat ctaatcctag ata 331633DNAartificial sequencechemically synthesized 16ttctgtcgtt ccacatcttt tgctcccatg tat 331733DNAartificial sequencechemically synthesized 17agcgcaaccc ttatcgttag ttacattgtt taa 331834DNAartificial sequencechemically synthesized 18cgttagttac attgtttaac gagactgcta atgt 341934DNAartificial sequencechemically synthesized 19acgtgctaca atggccaata caaacagtag ccaa 342034DNAartificial sequencechemically synthesized 20ttgcagccct caatccgaac tgagaccaac tttt 342133DNAartificial sequencechemcially synthesized 21catagctgat tcgcgattac tagtgattcc agc 332233DNAartificial sequencechemically synthesized 22ttccaataaa ggttagcaac acgtttttaa ata 332333DNAartificial sequencechemically synthesized 23ttggacttga aacaataaca acttctcttc act 332433DNAartificial sequencechemically synthesized 24aagattactg gagagaaccc aggatcattt gga 332533DNAartificial sequencechemically synthesized 25cagtgggcag actatgtctt acctttgatt gta 332634DNAartificial sequencechemically synthesized 26ttatccttag tgttactttg ggattaacga ttgg 342735DNAartificial sequencechemically synthesized 27caatgcacag aaacaaaaag gcattacaag caggg 352833DNAartificial sequencechemically synthesized 28tctgattgca aagttttgct gaccatcaag gta 332931DNAartificial sequencechemically synthesized 29ctctaccgtt gttatcatac cttctgattg c 313033DNAartificial sequencechemically synthesized 30ttctgttaat gatctcttta aagacactac caa 333134DNAartificial sequencechemically synthesized 31cttaggagcg ttagagatcc ctgttctgtt aatg 343234DNAartificial sequencechemcially synthesized 32cttgttttaa cttcttagga gcgttagaga tccc 343334DNAartificial sequencechemically synthesized 33ttactggagg ttttggtggg gttttaggag ttgg 343434DNAartificial sequencechemically synthesized 34ctcctccggg tgatgctttt cctagattat tctc 343532DNAartificial sequencechemically synthesized 35ctaaccctgc gctcctgcct cgatgggtgg ag 323633DNAartificial sequencechemically synthesized 36aagtcaggtg gagcgaggct agctggcccg att 333734DNAartificial sequencechemically synthesized 37tccttttcca actacccatg actcagcttc tccc 343833DNAartificial sequencechemically synthesized 38caccatgcca cagccaccac acctctgcgg gga 333933DNAartificial sequencechemically synthesized 39ccaccaccag aggggccatt ttgcggtgga aat 334021DNAartificial sequencechemically synthesized 40gcttgttgga aacaaatctg a 214122DNAartificial sequencechemically synthesized 41tcgaacattt tttaaaacca gg 224243DNAartificial sequencechemically synthesized 42gatcgcccag acaatgctcc taatctccaa gcttaagact tca 434339DNAartificial sequencechemically synthesized 43aaccaatccc gggcattgat aaaaacggat gcgatgaac 394421DNAartificial sequencechemically synthesized 44aaagtgcata aacttctgag g 214521DNAartificial sequencechemically synthesized 45ctaaaaaaaa tcaatgcccg g 214648DNAartificial sequencechemically synthesized 46tgtttccaac aagctaccat ttcttataat cctcttttct gtctgacg 484743DNAartificial sequencechemcially sytnhesized 47aatctccaag cttaagactt cagagattgg ttgatcgccc aga 434823DNAartificial sequencechemically synthesized 48tctaaagaca aaaaagatcc tcg 234919DNAartificial sequencechemically synthesized 49tgtgatgggt aaagggatt 195050DNAartificial sequencechemically synthesized 50gcatgaaaag cttctcctta ttcgaatgat ctacaagtat gtttgttgag 505145DNAartificial sequencechemically synthesized 51ccaataggat tcttggcgaa ttttttgcag caagaaatgt cgtta 455225DNAartificial sequencechemically synthesized 52cgactatttt cttgtttaga aggtt 255322DNAartificial sequencechemically synthesized 53gaaaagattg gtctattgtc ct 225446DNAartificial sequencechemically synthesized 54agcagcaagc tatatttcct taacagctat agcgactatt ccttga 465547DNAartificial sequencechemically synthesized 55gtcttggcag aggaaacttt tttaatggat atgaatctgc aagagtt 475619DNAartificial sequencechemically synthesized 56gattcctaaa caggatgac 195718DNAartificial sequencechemically synthesized 57tcgcatctag gattagat 185820DNAartificial sequencechemically synthesized 58agattcctaa acaggatgac 205921DNAartificial sequencechemically synthesized 59cgcatctagg attagattat g 216020DNAartificial sequencechemically synthesized 60aatatcatct ttgcggttgc 206121DNAartificial sequencechemically synthesized 61tctacaagag tacatcggtc a 216239DNAartificial sequencechemically synthesized 62tcgagcaacc gctgtgacga ccttcattat gtcggagtc 396339DNAartificial sequencechemically synthesized 63gcagcttgta gtcctgcttg agtcttcgta actcgctcc 396418DNAartificial sequencechemcially synthesized 64tacaaacgcc tagggtgc 186518DNAartificial sequencechemically synthesized 65cgggcgattt gccttaac 186619DNAartificial sequencechemically synthesized 66gcagttgcag gaagagatc 196722DNAartificial sequencechemically synthesized 67gtcatcttga agaagatacg aa 226849DNAartificial sequencechemically synthesized 68ggacttttgg attcgggata aaatgctgat attctgattg catgtatcg 496939DNAartificial sequencechemically synthesized 69ggaggactgg gcatttcttc atggaatact ccaggtcgc 397017DNAartificial sequencechemically synthesized 70agcgttggag ccacctc 177123DNAartificial sequencechemically synthesized 71gaaaacatgc agcacgtttt gca 237220DNAartificial sequencechemically synthesized 72caatagcgtt ggagccacct 207320DNAartificial sequencechemically synthesized 73aacatgcagc acgttttgca 207420DNAartificial sequencechemically synthesized 74caagatgacg atggacaagt 207522DNAartificial sequencechemically synthesized 75ccagataagt taacgatgac ga 227640DNAartificial sequencechemically synthesized 76ggctcgtcct gactcatgct ccgctggatt agattatcct 407741DNAartificial sequencechemically synthesized 77ccgatgaaga ggcgttacga ggagcatgtg aagactccaa t 417819DNAartificial sequencechemically synthesized 78catgatctgg cccaactga 197922DNAartificial sequencechemically synthesized 79tcctgcttac tagaaatgag gg 228022DNAartificial sequencechemically synthesized 80attggttaac ttacctagtg gc 228121DNAartificial sequencechemically synthesized 81acttcttagg agcgttagag a 218241DNAartificial sequencechemically synthesized 82gacatagtct gcccactggt tgatcctcaa acccaacagt t 418341DNAartificial sequencechemically synthesized 83aggcattaca agcagggttt gaaagacact accaactgct t 418422DNAartificial sequencechemically synthesized 84aaagggttga aagacagttt gg 228519DNAartificial sequencechemically synthesized 85aaggttgatg tcttgacca 198621DNAartificial sequencechemically synthesized 86caccttacca gtaactgaac t 218718DNAartificial sequencechemically synthesized 87aaccctgctt gtaatgcc 188844DNAartificial sequencechemically synthesized 88ttaagcggat tgaagcttga tctgtctatg accagtatgt acca 448942DNAartificial sequencechemically synthesized 89cccaacagtt tatcccggta ctaaggtaag acatagtctg cc 429022DNAartificial sequencechemically synthesized 90gccactaggt aagttaacca at 229120DNAartificial sequencechemically synthesized 91aatgcatcaa gtacaggtcc 209221DNAartificial sequencechemically synthesized 92caccttacca gtaactgaac t 219318DNAartificial sequencechemically synthesized 93aaccctgctt gtaatgcc 189444DNAartificial sequencechemically synthezised 94ttaagcggat tgaagcttga tctctatgac cagtatgtac cact 449542DNAartificial sequencechemically synthesized 95cccaacagtt tatcccggta ctaaggtaag acatagtctg cc 429622DNAartificial sequencechemically synthesized 96gccactaggt aagttaacca at 229720DNAartificial sequencechemically synthesized 97aatgcatcaa gtacaggtcc 209821DNAartificial sequencechemically synthesized 98caccttacca gtaactgaac t 219918DNAartificial sequencechemically synthesized 99aaccctgctt gtaatgcc 1810044DNAartificial sequencechemically synthesized 100ttaagcggat tgaagcttga tcgtctatga ccagtatgta ccac 4410142DNAartificial sequencechemically synthesized 101cccaacagtt tatcccggta ctaaggtaag acatagtctg cc 4210222DNAartificial sequencechemically synthesized 102gccactaggt aagttaacca at 2210320DNAartificial sequencechemically synthesized 103aatgcatcaa gtacaggtcc 2010420DNAartificial sequencechemically synthesized 104gatcctcaaa cccaacagtt 2010520DNAartificial sequencechemically synthesized 105ttaggagttg gtttggttgg 2010639DNAartificial sequencechemically synthesized 106gacatagtct gcccactggt ttgcatcaag tacaggtcc 3910743DNAartificial sequencechemically synthesized 107aggcattaca agcagggttt gaacttctta ggagcgttag aga 4310822DNAartificial sequencechemically synthesized 108aaagggttga aagacagttt gg 2210920DNAartificial sequencechemically synthesized 109aaggttgatg tcttgaccaa 2011022DNAartificial sequencechemically synthesized 110tgtctatgac cagtatgtac ca 2211118DNAartificial sequencechemically synthesized 111aaccctgctt gtaatgcc 1811244DNAartificial sequencechemically synthesized 112actgttgggt ttgaggatct ttattggtta acttacctag tggc 4411342DNAartificial sequencechemically synthesized 113cccggtacta aatgcatcaa gtaaggtaag acatagtctg cc 4211422DNAartificial sequencechemically synthesized 114ttaagcggat tgaagcttga tc 2211522DNAartificial sequencechemically synthesized 115ccaaactgtc tttcaaccct tt 2211621DNAartificial sequencechemically synthesized 116caccttacca gtaactgaac t 2111718DNAartificial sequencechemically synthesized 117aaccctgctt gtaatgcc 1811844DNAartificial sequencechemically synthesized 118ttacctttaa gcggattgaa gctgaccagt atgtaccact attg 4411942DNAartificial sequencechemically synthesized 119cccaacagtt tatcccggta ctaaggtaag acatagtctg cc 4212022DNAartificial sequencechemically synthesized 120gatcaaagcc actaggtaag tt 2212120DNAartificial sequencechemically synthesized 121aatgcatcaa gtacaggtcc 2012222DNAartificial sequencechemically synthesized 122ctatgaccag tatgtaccac ta 2212318DNAartificial sequencechemically synthesized 123aaccctgctt gtaatgcc 1812443DNAartificial sequencechemically synthesized 124actgttgggt ttgaggatct ttttggttaa cttacctagt ggc 4312542DNAartificial sequencechemically synthesized 125cccggtacta aatgcatcaa gtaaggtaag acatagtctg cc 4212622DNAartificial sequencechemically syntesized 126ttaagcggat tgaagcttga tc 2212722DNAartificial sequencechemically synthesized 127ccaaactgtc tttcaaccct tt 2212820DNAartificial sequencechemically synthesized 128tgaccagtat gtaccactat 2012918DNAartificial sequencechemically synthesized 129aaccctgctt gtaatgcc 1813043DNAartificial sequencechemically synthesized 130actgttgggt ttgaggatct tttggttaac ttacctagtg gct 4313142DNAartificial sequencechemically synthesized 131cccggtacta aatgcatcaa gtaaggtaag acatagtctg cc

4213222DNAartificial sequencechemically synthesized 132ttaagcggat tgaagcttga tc 2213322DNAartificial sequencechemically synthesized 133ccaaactgtc tttcaaccct tt 2213422DNAartificial sequencechemically synthesized 134tgaccagtat gtaccactat tg 2213518DNAartificial sequencechemically synthesized 135aaccctgctt gtaatgcc 1813642DNAartificial sequencechemically synthesized 136actgttgggt ttgaggatct ttgttaactt acctagtggc tt 4213742DNAartificial sequencechemically synthesized 137cccggtacta aatgcatcaa gtaaggtaag acatagtctg cc 4213822DNAartificial sequencechemically synthesized 138ttaagcggat tgaagcttga tc 2213922DNAartificial sequencechemically synthesized 139ttaagcggat tgaagcttga tc 2214020DNAartificial sequencechemically synthesized 140gaccagtatg taccactatt 2014118DNAartificial sequencechemically synthesized 141aaccctgctt gtaatgcc 1814243DNAartificial sequencechemically synthesized 142actgttgggt ttgaggatct ttggttaact tacctagtgg ctt 4314342DNAartificial sequencechemically synthesized 143cccggtacta aatgcatcaa gtaaggtaag acatagtctg cc 4214422DNAartificial sequencechemically synthesized 144ttaagcggat tgaagcttga tc 2214522DNAartificial sequencechemically synthesized 145ccaaactgtc tttcaaccct tt 2214619DNAartificial sequencechemically synthesized 146cgtgaacgat gaaggtctt 1914722DNAartificial sequencechemcially synthesized 147accacactct agactgatag tt 2214840DNAartificial sequencechemically synthesized 148gcgactgctg gcacatagtt aagaatgact ctagcaggca 4014942DNAartificial sequencechemically synthesized 149acataggtcg caagcgttat ccctgccttt aacaccagac tt 4215020DNAartificial sequencechemically synthesized 150gtacagtcaa actccagcca 2015122DNAartificial sequencechemically synthesized 151ggatttattg ggcgtaaagc aa 2215219DNAartificial sequencechemically synthesized 152cgtgaacgat gaaggtctt 1915322DNAartificial sequencechemically synthesized 153accacactct agactgatag tt 2215439DNAartificial sequencechemically synthesized 154gcgactgctg gcacatagta atgactctag caggcaatg 3915542DNAartificial sequencechemically synthesized 155acataggtcg caagcgttat ccctgccttt aacaccagac tt 4215620DNAartificial sequencechemically synthesized 156tggtacagtc aaactccagc 2015722DNAartificial sequencechemically synthesized 157ggatttattg ggcgtaaagc aa 2215819DNAartificial sequencechemically synthesized 158cgtgaacgat gaaggtctt 1915922DNAartificial sequencechemically synthesized 159accacactct agactgatag tt 2216042DNAartificial sequencechemically synthesized 160gctggcacat agttagtcgt cagaagaatg actctagcag gc 4216142DNAartificial sequencechemically synthesized 161acataggtcg caagcgttat ccctgccttt aacaccagac tt 4216220DNAartificial sequencechemically synthesized 162gtacagtcaa actccagcca 2016322DNAartificial sequencechemically synthesized 163ggatttattg ggcgtaaagc aa 2216419DNAartificial sequencechemically synthesized 164cgtgaacgat gaaggtctt 1916522DNAartificial sequencechemically synthesized 165accacactct agactgatag tt 2216641DNAartificial sequencechemically synthesized 166gcgactgctg gcacatagtt agaatgactc tagcaggcaa t 4116742DNAartificial sequencechemically synthesized 167acataggtcg caagcgttat ccctgccttt aacaccagac tt 4216820DNAartificial sequencechemically synthesized 168tggtacagtc aaactccagc 2016922DNAartificial sequencechemically synthesized 169ggatttattg ggcgtaaagc aa 2217020DNAartificial sequencechemically synthesized 170cattactgac gcttaggctt 2017120DNAartificial sequencechemically synthesized 171gccaaggatg tcaagtctag 2017241DNAartificial sequencechemically synthesized 172cttcactacc gaagggatcg ccctagtagt ccacaccgta a 4117339DNAartificial sequencechemically synthesized 173gcctgggtag tacattcgca aaacatgctc caccacttg 3917422DNAartificial sequencechemically synthesized 174tccgacagct agtatctatc gt 2217522DNAartificial sequencechemically synthesized 175tgaaactcaa acggaattga cg 2217619DNAartificial sequencechemically synthesized 176cgtgaacgat gaaggtctt 1917722DNAartificial sequencechemically synthesized 177accacactct agactgatag tt 2217837DNAartificial sequencechemically synthesized 178gcgactgctg gcacatagtg actctagcag gcaatgg 3717942DNAartificial sequencechemically synthesized 179acataggtcg caagcgttat ccctgccttt aacaccagac tt 4218022DNAartificial sequencechemically synthesized 180aaagtggtac agtcaaactc ca 2218122DNAartificial sequencechemically synthesized 181ggatttattg ggcgtaaagc aa 2218218DNAartificial sequencechemically synthesized 182caagtggtgg agcatgtt 1818319DNAartificial sequencechemically syntehsized 183tcccttcctt cctccaatt 1918439DNAartificial sequencechemcially synthesized 184cgacaaccat gcaccacctc tagacttgac atccttggc 3918542DNAartificial sequencechemically synthesized 185cagctcgtgt cgtgagatgt ttaactaacg ataagggttg cg 4218621DNAartificial sequencechemically synthesized 186gtcactcggt taacctccat t 2118718DNAartificial sequencechemically synthesized 187ggttaagtcc cgcaacga 1818820DNAartificial sequencechemically synthesized 188aatgactcta gcaggcaatg 2018922DNAartificial sequencechemically synthesized 189accacactct agactgatag tt 2219041DNAartificial sequencechemically synthesized 190cggataacgc ttgcgacctt aagtgacgac taactatgtg c 4119140DNAartificial sequencechemically synthesized 191aagcgcaggc ggattgaacc aatgcataca actgttaagc 4019219DNAartificial sequencechemically synthesized 192tgtattaccg cgactgctg 1919320DNAartificial sequencechemically synthesized 193agtctggtgt taaaggcagc 2019420DNAartificial sequencechemically synthesized 194aatgactcta gcaggcaatg 2019522DNAartificial sequencechemically synthesized 195accacactct agactgatag tt 2219638DNAartificial sequencechemically synthesized 196gataacgctt gcgacctaag tgacgactaa ctatgtgc 3819740DNAartificial sequencechemically synthesized 197aagcgcaggc ggattgaacc aatgcataca actgttaagc 4019819DNAartificial sequencechemically synthesized 198tgtattaccg cgactgctg 1919920DNAartificial sequencechemically synthesized 199agtctggtgt taaaggcagc 2020018DNAartificial sequencechemically sythesized 200caagtggtgg agcatgtt 1820120DNAartificial sequencechemically synthesized 201gtttgcagcc ctagacataa 2020240DNAartificial sequencechemically synthesized 202cgacacgagc tgacgacaac cttggcaaag ttatggaaac 4020341DNAartificial sequencechemically synthesized 203tgggttaagt cccgcaacgc caatttacat tagcagtctc g 4120418DNAartificial sequencechemically synthesized 204catgcaccac ctgtcact 1820522DNAartificial sequencechemically synthesized 205cgcaaccctt atcgttagtt ac 2220621DNAArtificial sequencechemically synthesized 206cgcataagaa ctttagttcg c 2120719DNAartificial sequencechemically synthesized 207aagaccttca tcgttcacg 1920843DNAartificial sequencechemically synthesized 208tagctacacg tcattgcctt ggagggttcg ttatttgatg agg 4320940DNAartificial sequencechemically synthesized 209cacaatggga ctgagacacg gagctttcgc tcattgtgaa 4021022DNAartificial sequencechemically synthesized 210cctaccaact agctgatatg gc 2221118DNAartificial sequencechemically synthesized 211tactcctacg ggaggcag 1821218DNAartificial sequencechemically synthesized 212tgggtgtgaa ccatgaga 1821319DNAartificial sequencechemically synthesized 213agtccttcca cgataccaa 1921439DNAartificial sequencechemically synthesized 214tccatagggt gccaggctgt atgacaacag cctcaagat 3921542DNAartificial sequencechemically synthesized 215ctttctttgc agcaatgcct ccagttgtca tggatgacct tg 4221619DNAartificial sequencechemically synthesized 216ctgccttcct cacctgatg 1921718DNAartificial sequencechemically synthesized 217tgcaccacca actgctta 1821816DNAartificial sequencechemically synthesized 218ccccaaaggc caggct 1621919DNAartificial sequencechemically synthesized 219agaagggatg ggagagagc 1922042DNAartificial sequencechemcially synthesized 220ggaatgggga gaagggcagg ttaaatgtca ccgggaggat tg 4222141DNAartificial sequencechemically synthesized 221cggaaaccag atctcccacc ggctacagaa aggtcagcag c 4122218DNAartificial sequencechemically synthesized 222atcaagtggg gcgatgct 1822319DNAartificial sequencechemically synthesized 223gggcagagat gatgaccct 1922439DNAartificial sequencechemically synthesized 224gcactcaccc cagccttctc gctgagtacg tcgtggagt 3922540DNAartificial sequencechemcially synthesized 225aagctgactc agccctgcaa accctgcaaa tgagcctaca 4022618DNAartificial sequencechemically synthesized 226gttgacccga ccccaaag 1822718DNAartificial sequencechemically synthesized 227aagggatggg agagagcc 1822839DNAartificial sequencechemically synthesized 228cggaatgggg agaagggcag atgtcaccgg gaggattgg 3922941DNAartificial sequencechemically synthesized 229cggaaaccag atctcccacc gccagctaca gaaaggtcag c 41

Claims

1. A method for detection of nucleic acid targets from biological samples and body fluids comprising the steps of: a) sample pretreatment comprising cell lysis and release of nucleic acid targets in biological samples and body fluids; b) amplification of nucleic acid(s); c) detection of amplification product(s), wherein lytic peptides are used to release nucleic acid targets in biological samples or body fluids.

2. The method according to claim 1, wherein detergents are used to release nucleic acid targets in biological samples or body fluids.

3. The method according to claim 1, wherein combination of lytic peptides and detergents are used to release nucleic acid targets in biological samples or body fluids.

4. The method according to claim 1, wherein one or more specific target based sequences are amplified.

5. The method according to claim 1, wherein sample solution obtained during the step a) is directly subjected for further amplification procedure.

6. The method according to claim 1, wherein qualitative and quantitative detection is performed with crude sample solution.

7. The method according to claim 1, wherein the Chlamydia trachomatis nucleic acid target(s) with the use of specific target region provided in Table 1 is detected.

8. The method according to claim 1, wherein the Mycoplasma genitalium nucleic acid target(s) with the use of specific target region provided in Table 1 is detected.

9. The method according to claim 1, wherein the Chlamydia trachomatis nucleic acid target(s) with the use of specific primer(s) and/or its labeled derivative(s) sequences provided in Table 2 and 3 is detected.

10. The method according to claim 1, wherein the Mycoplasma genitalium nucleic acid target(s) with the use of specific primer(s) and/or its labeled derivative(s) sequences provided in Table 2 and 3 is detected.

11. The method according to claim 1, wherein the human genomic GAPDH target is used for detection as an internal validation and platform assessing technique.

12. The method according to claim 1, wherein the human genomic GAPDH target is used for detection as an internal validation and platform assessing technique with specific primer(s) and/or its labeled derivative(s) sequences provided in Tables 2, 3.

13. A molecular diagnostics method of Chlamydia trachomatis, wherein the TRPB gene is used as molecular diagnostics target.

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